Abstract:
:Cutaneous melanoma is an increasingly common and potentially lethal malignancy of melanocytes, the melanin producing cells normally located in the basal layer of the skin epidermis. Despite major advances in cancer chemotherapeutics and immunotherapy, the success in treating metastatic melanoma remains poor. The notion that cancer stem cells (CSCs) play a key role in melanoma progression is well received. Therefore, isolating and characterizing CSCs is of critical importance for designing new therapeutic strategies that target this unique tumor initiating cell sub-population. In this work, we present a simple in vitro method, employing cell culture on polydimethylsiloxane (PDMS) and transfer back onto standard tissue culture plate, to enrich a non-adherent spheroid (NA/S) forming and an adherent monolayer (AM) cell sub-populations from the tumorigenic WM115 melanoma cell line. The phenotypes of the morphologically distinct NA/S and AM sub-populations were further characterized by quantifying the expression of stem cell markers, CD20 and CD271. Flow cytometric analysis found 2.32% of the cells in the NA/S sub-population were CD20+ CD271+ whereas only 0.27% of the cells in the AM sub-population were CD20+ CD271+. When the NA/S sub-population was cultured back onto PDMS it resulted in the further enrichment of CD20+ CD271+ cells to 14.7%. We used microbubble arrays to quantify the in vitro clonogenic potential of the NA/S and AM cell sub-populations. Microbubbles are spherical cavities, ~160 μm in diameter with 60 μm circular openings, formed in PDMS using the gas expansion molding (GEM) process. Cells from each sub-population were seeded, under limiting dilution conditions, onto separate arrays containing 1215 microbubble wells. After five days in culture, wells seeded with 1, 2, 3 and >3 cells per microbubble well were inspected for cell proliferation. The Extreme Limiting Dilutions Analysis (ELDA) determined a ~58% clonal survival (1 in every 1.72 cells) for the NA/S sub-population and ~25% clonal survival (1 in every 3.93 cells) for the AM sub-population (= 176, p = 4.41e(-40)). These findings taken together add to the existing evidence that melanoma cells propagating as non-adherent/spheroids represent a more aggressive phenotype due to the greater presence of tumor initiating cells.
journal_name
Biomaterialsjournal_title
Biomaterialsauthors
Chandrasekaran S,DeLouise LAdoi
10.1016/j.biomaterials.2011.08.056subject
Has Abstractpub_date
2011-12-01 00:00:00pages
9316-27issue
35eissn
0142-9612issn
1878-5905pii
S0142-9612(11)01004-0journal_volume
32pub_type
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